Apparatus and method for segmented thermal cycler

ABSTRACT

The present invention relates to a thermal cycler for the carrying out of chemical or biological reactions, such as PCR or other nucleic acid amplification reactions, that is segmented with a plurality of reaction vessel receiving elements. The reaction vessel receiving elements are thermally isolated from each other and provide an airtight seal to prevent liquids or moisture from penetrating below the reaction vessel receiving elements. The reaction vessel receiving elements have several recesses arranged in a pattern to receive the reaction vessels of a single standard microtiter plate and the segmented thermal cycler has a system for independently heating and cooling each of the reaction vessel receiving elements.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 14/617,831filed Feb. 9, 2015, which is a continuation of U.S. application Ser. No.12/617,568 filed Nov. 12, 2009, which claims a priority under 35 U.S.C.§ 119(e) from U.S. Provisional Application No. 61/114,902 filed Nov. 14,2008, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a thermal cycler for the carrying outof chemical or biological reactions, such as PCR or other nucleic acidamplification reactions, the thermal cycler is segmented in that it hasa plurality of reaction vessel receiving elements separated andthermally isolated from each other for receiving reaction vessels,wherein the reaction vessel receiving elements have several recessesarranged in a pattern to receive the reaction vessels of a singlestandard microtiter plate. The segmented thermal cycler has a system forindependently heating and cooling each of the reaction vessel receivingelements.

INTRODUCTION

Testing of biological or chemical samples often requires a device forrepeatedly subjecting multiple samples though a series of temperaturecycles. Such devices are described as thermal cyclers or thermocyclingdevices and are used to generate specific temperature cycles, i.e. toset predetermined temperatures in the reaction vessels and to maintainpredetermined intervals of time, sometimes called protocols. Often timesit is desirable to thermocycle different samples in a single vesselarray through different protocols by varying temperature, time, and/ornumber of cycles, where these experiments can be carried outsimultaneously.

For example, such tests can used to determine the optimal denaturingtemperature, the optimal annealing temperature, and the optimalelongation temperature of a PCR reaction. To achieve this, the samereaction mixture is poured into the individual reaction vessels, and thetemperature cycles necessary to perform the PCR reaction are executed.Such a temperature cycle comprises the heating of the reaction mixtureto the denaturing temperature, which usually lies in the range 90°-95°C., cooling to the annealing temperature, which is usually in the range40°-60° C. and heating to the elongation temperature, which is usuallyin the range 70°-75° C. If desired, the time of each cycle can also bevaried. A cycle of this kind is repeated several times, leading toamplification of a predetermined DNA sequence. The annealingtemperature, at which the primer is added, has a powerful influence onthe result. However the elongation temperature too can have beneficialor adverse effects on the result. At a higher elongation temperature,the addition of the bases is accelerated, with the probability of errorsincreasing with higher temperature. In addition, the life of thepolymerase is shorter at a higher elongation temperature. Anotherimportant parameter for the success of a PCR reaction is the differentresidence volumes spread over different reaction vessels. Problems arisewith conventional devices as these parameters can not be varied in onetest series for an individual reaction vessel holder. To test differentresidence volumes, several test series are required and are performedeither consecutively in one thermocycling device or simultaneously inseveral thermocycling devices.

Historically, only temperature could be varied during thermal cyclingusing a gradient thermal cycler that can create a temperature gradientand/or a gradient block as described in, for example, U.S. Pat. Nos.5,525,300 and 7,074,367. The disclosed devices have a single block andcreate a gradient of temperatures and then try to designate differenttemperature for samples.

The next generation of thermal cyclers adopted a markedly differentapproach. Rather than a single gradient block, these new thermal cyclersembodied a plurality of blocks or reaction vessel receiving elementsthat are isothermal where each reaction vessel receiving element isindependently controlled and can be programmed with different protocols,while having the proximity to be arranged in a pattern to receive thereaction vessels of a single vessel array of a standard format asdescribed in, for example, U.S. Pub. No. 2006-0228268. Different butpredetermined temperatures are set for each of the reaction vesselreceiving elements. After completion of the cycles it is possible todetermine, with the aid of the reaction products, those temperatures atwhich the PCR reaction will give the user the optimal result. Here theresult may be optimized e.g. in respect of product volume or alsoproduct quality. The present invention is an improvement to such athermal cycler, by providing a different means for thermal isolation ofthe reaction vessel receiving elements and a seal to reduce the abilityof liquids and moisture from penetrating between the reaction vesselreceiving elements.

SUMMARY OF THE INVENTION

According to various embodiments, the present teachings describe athermal cycler for processing biological or chemical samples with aplurality of reaction vessel receiving elements configured to receiveone standard microtiter plate, a plurality of thermoelectric coolingdevices (TEC) disposed to correspond to each of the plurality ofreaction vessel receiving elements, wherein the TEC provides heating andcooling, a drip pan positioned above the TECs and framing the pluralityof reaction vessel receiving elements, a single gasket to seal theplurality of reaction vessel receiving elements, wherein the gasket hasa convex construction, and a clamp to provide lateral force to compressthe gasket between the reaction vessel receiving elements, wherein thegasket forms an airtight seal between each of the plurality of reactionvessel receiving elements and between the drip pan and the plurality ofreaction vessel receiving elements to isolate the plurality of TECs fromenvironmental conditions above the drip pan and the plurality ofreaction vessel receiving elements, and wherein the gasket is composedof non-thermally conducting material and separates adjacent reactionvessel receiving elements to provide thermal isolation between adjacentreaction vessel receiving elements.

According to various embodiments, the present teachings describe amethod for processing biological or chemical samples includingpositioning a single standard microtiter plate on a plurality ofreaction vessel receiving elements of a thermal cycler, independentlyheating and cooling the plurality of reaction vessel receiving elementswith a plurality of thermoelectric cooling devices (TEC), sealing thearea below the plurality of reaction vessel receiving elements with adrip pan, a gasket, and a clamp, wherein the gasket has a convex portionand the clamp provides a lateral force to compress the gasket betweenthe reaction vessel receiving elements to form an airtight seal betweeneach of the plurality of reaction vessel receiving elements, andthermally isolating adjacent reaction vessel receiving elements byconstructing the gasket from a non-thermally conducting material.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a top view of a portion of a device according to theinvention for carrying out chemical or biological reactions inaccordance with an exemplary embodiment showing the reaction vesselreceiving elements, gasket, and drip pan;

FIG. 2 depicts a perspective view of a reaction vessel receivingelement, according to various embodiments;

FIG. 3 depicts a cross-sectional view of the device of along the axisshown in FIG. 1;

FIG. 4 depicts a magnified view of a portion of FIG. 3;

FIGS. 5 and 6 depict a perspective view and a side view of a gasket,according to various embodiments;

FIGS. 7 and 8 depict cross-sectional views of the gasket in FIG. 6 alongthe axes A-A and B-B, respectively;

FIGS. 9 and10 depict magnified views of portions of FIG. 7;

FIGS. 11A and 11B depict top and bottom perspective views of the clamp,according to various embodiments;

FIG. 12 depicts an exploded view of the bottom portion of a thermalcycler, according to various embodiments;

FIG. 13 depicts an exploded view of the top portion of a thermal cycler,according to various embodiments; and

FIG. 14 depicts an exploded view of a thermal cycler, according tovarious embodiments.

DESCRIPTION OF THE EMBODIMENTS

In the following description, reference is made to the accompanyingdrawings that form a part thereof, and in which are shown by way ofillustration specific exemplary embodiments in which the invention maybe practiced. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention and it is tobe understood that other embodiments may be utilized and that changesmay be made without departing from the scope of the invention. Thefollowing description is, therefore, not to be taken in a limited sense.

As used herein, the term “microtiter plate” is also known as a “sampleplate,” “microtitration plate,” and “microplate” interchangeably andrefers to a multi-welled sample receptacle for testing of chemical andbiological samples. Microplates can have wells that are conical,cylindrical, rectilinear, tapered, and/or flat-bottomed in shape, andcan be constructed of a single material or multiple materials. Astandard microtiter plate conforms to SBS Standards. Microtiter platescan be open-faced (e.g. closed with a sealing film or caps) orclose-chambered (e.g. microcard as described in U.S. Pat. No.6,825,047). Open-faced microtiter plates can be filled, for example,with pipettes (hand-held, robotic, etc.) or through-hole distributionplates. Close-chambered microtiter plates can be filled, for example,through channels or by closing to form the chamber. Examples of standardmicrotiter plates have the following number of wells or chambers: 24,48, 96, 384, or 1536.

FIGS. 1 to 14 depict exemplary embodiments of methods and systems thatinclude a plurality of reaction vessel receiving elements thermallydecoupled and each segment assigned a thermoelectric cooler (TEC), alsoPeltier cooler, which may be actuated independently.

By this means the individual reaction vessel receiving element of thedevice may be set to different temperatures independently of oneanother. This makes it possible not only to set different temperaturelevels in the segments, but also for them to be held for varying lengthsof time or altered at different rates of change. The device according tothe invention thus permits optimization of all physical parameterscritical for a PCR process, while the optimization process may becarried out on a single reaction vessel receiving element in which asingle standard microtiter plate may be inserted.

With the device according to the invention it is therefore also possibleto optimize the residence times and rates of temperature change withouthaving to distribute the reaction mixture over different microtiterplates for this purpose. Moreover, it is also possible to optimize themixture volume by varying the mixture volume over different reactionvessel segments. The thermal cycling device according to the inventionis in particular suitable for optimizing the multiplex PCR process, inwhich several different primers are used.

According to various embodiments, FIG. 1 shows a top view of a portionof a device according to the invention for carrying out chemical orbiological reactions in accordance with an exemplary embodiment showingthe reaction vessel receiving elements 10, gasket 20, and drip pan 30.

According to various embodiments, FIGS. 2-4 shows the recesses of areaction vessel receiving element 10 and how a plurality reaction vesselreceiving elements 10 are configures to be surrounded by gasket 20 anddrip pan 30 to form an airtight seal between each of the plurality ofreaction vessel receiving elements and between the drip pan and theplurality of reaction vessel receiving elements 10 to isolate theplurality of TECs 40 below each reaction vessel receiving element 10from environmental conditions above the drip pan 30 and the plurality ofreaction vessel receiving elements 10. This is achieved by sealing thearea below the plurality of reaction vessel receiving elements 10 with agasket 20 that has a convex construction as shown in FIGS. 4-10. Thegasket 20 has a convex portion with ribs 50 that provide contact theplurality of reaction vessel receiving elements 10. The clamp 60 asshown in FIGS. 11A and 11B provides a lateral force to compress thegasket between the reaction vessel receiving elements 10 to form anairtight seal between each of the plurality of reaction vessel receivingelements 10.

According to various embodiments, FIG. 12 shows an exploded view of thebottom portion of a thermal cycler with the clamp 60 providing lateralforce to the plurality of reaction vessel receiving elements 10 tocompress gasket 20. Each reaction vessel receiving element 10 is coupledto a TEC 40 via a thermally conductive material 70. The TECs 40 arealigned by frame 80 and powered by a printed circuit board 90. The TECs40 are coupled to heat sink 100 by thermally conductive material 70. Theclamp 60, in addition to providing lateral force to the plurality ofreaction vessel receiving elements 10, can provide vertical clamping ofthe plurality of reaction vessel receiving elements 10 to the TECs 40and heat sink 100. The TECs can be powered by current flow from aplurality of power amplifiers. The power amplifiers can be coupled tothe TEC via actual leads or can be coupled via infrared connection topermit tighter placement of TECs and reaction vessel receiving elements10. According to various embodiments, the present teachings contemplateone or more temperature sensors disposed in each reaction vesselreceiving element in conjunction with each TEC. According to variousembodiments, the present teachings contemplate a heating elementdisposed in each reaction vessel receiving element, wherein the heatingelement provides fine heating to a control temperature. According tovarious embodiments, the present teachings contemplate a plurality ofpower amplifiers, and a switch for each of the plurality of reactionvessel receiving elements to direct a current flow from the plurality ofpower amplifiers to the TEC. According to various embodiments, thepresent teachings contemplate the TECs corresponding to each of thereaction vessel receiving elements are integrated into a single unit. Invarious embodiments, the TEC can include dicing to segment portionsaccording to the reaction vessel receiving elements.

According to various embodiments, the present teachings contemplate areaction vessel receiving elements comprises a flat surface sampleblock.

According to various embodiments, FIG. 13 shows an exploded view of thetop portion of a thermal cycler with drip pan 30 and the heated lid,including heated platen 110, springs 120 to provide a downward force topress the heated platen 110 on the microtiter plate, cover 130 toenclose the heated platen 110 and springs 120, and locking handlemechanism 140 to close, lower, and lock the heated lid in place over themicrotiter plate sitting in the plurality of reaction vessel receivingelements 10.

According to various embodiments, FIG. 14 shows a thermal cyclerinstrument with the exploded view of FIG. 12 as bottom portion 150, andthe exploded view of FIG. 13 as top portion 160 with additionaldisplay/touch screen 170 and electrical housing 180.

The reaction vessel receiving elements 10 are not influenced by theother reaction vessel receiving elements 10, and their temperature maybe set completely independently of the other reaction vessel receivingelements 10. By this means it is possible to run quite differenttemperature cycles on the individual reaction vessel receiving elements10, with one of the reaction vessel receiving element 10 for exampleheated up to the denaturing temperature and another held at theannealing temperature. Thus it is possible for the residence times, i.e.the intervals of time for which the denaturing temperature, theannealing temperature and the elongation temperature are held, also therates of temperature change, to be set as desired, and runsimultaneously on the individual reaction vessel receiving elements 10.In this way it is possible to optimize not only the temperatures, butalso the residence times, mixture volume, and the rates of temperaturechange. According to various embodiments, the present teachings providefor a method of annealing samples in a first portion of the microtiterplate at a first annealing temperature by cooling a first reactionvessel receiving element, and annealing samples in a second portion ofthe microtiter plate at a second annealing temperature by cooling asecond reaction vessel receiving element, wherein the second annealingtemperature is not equal to the first annealing temperature. Accordingto various embodiments, the present teachings provide for a method ofelongating samples in a first portion of the microtiter plate at a firstelongation temperature by heating a first reaction vessel receivingelement, and elongating samples in a second portion of the microtiterplate at a second elongation temperature by heating a second reactionvessel receiving element, wherein the second elongation temperature isnot equal to the first elongation temperature. According to variousembodiments, the present teachings provide for a method of repeating fora first number of cycles at least one of the steps of denaturing,annealing, and elongating samples in a first portion of the microtiterplate corresponding to a first reaction vessel receiving element, andrepeating for a second number of cycles at least one of the steps ofdenaturing, annealing, and elongating samples in a second portion of themicrotiter plate corresponding to a second reaction vessel receivingelement, wherein the first number of cycles is not equal to the secondnumber of cycles. According to various embodiments, the presentteachings provide for a method where a rate of cooling of a firstreaction vessel receiving element is not equal to the rate of cooling ofa second reaction vessel receiving element, and/or a rate of heating ofa first reaction vessel receiving element is not equal to the rate ofheating of a second reaction vessel receiving element. According tovarious embodiments, the present teachings provide for a method wherethe samples in a first reaction vessel receiving element have adifferent volume than the samples in a second reaction vessel receivingelement. According to various embodiments, the present teachings providefor a method where a first reaction vessel receiving element is kept ata first residence time for annealing samples and a second reactionvessel receiving element is kept at a second residence time forannealing samples. According to various embodiments, the presentteachings provide for a method where a first reaction vessel receivingelement is kept at a first residence time for elongating samples and asecond reaction vessel receiving element is kept at a second residencetime for elongating samples

According to various embodiments, infrared sensors may for example beused as temperature sensors, located e.g. in the cover. With this sensorarrangement it is possible to sense the temperature of the reactionmixture directly. According to various embodiments, the heated platen110 can be sub-divided into segments that correspond to the reactionvessel receiving elements 10 so that each reaction vessel receivingelement 10 can have an independently controlled heated platen segmentthat can be varied in temperature. This variance can achieve optimizedheated lid conditions and/or provide heated platen segments to track thecycling of the reaction vessel receiving elements 10.

According to various embodiments, the reaction vessel receiving elements10 are made from a metal with good heat conducting properties, e.g.aluminum, copper, nickel, and/or silver. According to variousembodiments, reaction vessel receiving elements 10 can be machined,electroformed, or formed by metal injection molding (MIM). MIM cancombine the design freedom of plastic injection molding with theperformance of metal. MIM can be used with metals such as aluminum,copper, tungsten, and alloys thereof.

According to various embodiments, the gasket 20 is made from non-heatconducting materials or thermally insulating materials are eitherplastics or ceramics, for example silicone. The gasket 20 can also beflexible to provide an air-tight seal. An example of hardness to providesuch flexibility is durometer 30 shore A. According to variousembodiments, drip pan 30 can be formed of any suitable materialincluding but not limited to a thermoplastic. One of ordinary skill inthe art understands that the disclosed drip pans are exemplary and thatthe drip pan can be configured to receive the outer skirts of standardmicrotiter plates. According to various embodiments the drip pan 30 hasdemarcations for the standard microtiter plate, for example twelvecolumns (shown as columns 1-12) and 8 rows (shown as rows A-H) of wells.Although a 96 well sample plate with 16 wells in each reaction vesselreceiving element 10 is shown, one of ordinary skill in the art willunderstand that more or less wells can be included in each reactionvessel receiving element. One of ordinary skill in the art will alsounderstand that six reaction vessel receiving elements 10 is exemplaryand that more or less than six reaction vessel receiving elements 10 iscontemplated.

The invention is described above with the aid of embodiments with 96recesses for receiving a microtiter plate with 96 reaction vessels. Theinvention is not, however, limited to this number of recesses. Thus forexample the reaction vessel receiving element may also have 384 recessesto receive a corresponding microtiter plate. With regard to features ofthe invention not explained in detail above, express reference is madeto the claims and the drawing.

According to various embodiments, solid heated platen 110, can bereplaced with an apertured heated lid or a transparent heated lid topermit detection of samples in the standard microtiter plate duringamplification, e.g. real-time PCR. According to various embodiments, anexcitation light source and a detector can be included in cover 130 toprovide the mechanism for detection of sample held in the standardmicrotiter plate. In various embodiments, the reaction vessel receivingelement 10 can be combined with an excitation light source and adetector to provide monitoring of real-time PCR in samples in each ofthe reaction vessel receiving elements 10. Real-time PCR can bemonitored by detecting luminescence (for example, fluorescence,chemiluminescence, etc.) during the thermal cycling. In variousembodiments, the monitoring can be provided by imaging optics tooptically couple the samples in each of the reaction vessel receivingelements 10 with a detector, such as a CCD or PMT. Examples offluorescence detection with imaging optics embodiment are shown forexample at U.S. Pat. Nos. 7,295,316 and 7,423,750, both hereinincorporated by reference in their entirety. According to variousembodiments, a reaction vessel receiving element 10 is associated withdifferent regions on the detector, for example, a CCD. The detector canbe calibrated such that the regions corresponding to the assays that areperformed in each reaction vessel receiving element 10 so that detectionof the fluorescence is more efficient. According to various embodiments,the excitation light source can be one or more LEDs used to provideimproved illumination wavelength uniformity, light power outputuniformity, and minimal degradation of output over extended periods oftime. Further, LEDs operate at relatively low temperatures and requirelittle or no external cooling. According to various embodiments, thedetection optics can have sets of excitation filters, dichroic mirrors(beam-splitters), and emission filters. Alternatively, filter wheels onthe emission side and/or excitation side can provide differentexcitation and emission light patterns. According to variousembodiments, the present teachings describe a thermal cycler with anexcitation light source and a detector for monitoring real-time PCR thatcan include imaging optics optically coupling the samples in theplurality of reaction vessel receiving elements with a CCD or a scanninghead optically coupling the samples in the plurality of reaction vesselreceiving elements by movement over those segments.

The term “excitation light source” as used herein refers to a source ofirradiance that can provide excitation that results in fluorescentemission. Light sources can include, but are not limited to, LEDs,phosphor coated LEDs, organic LEDs (OLED), phosphorescent OLEDs(PHOLED), inorganic-organic LEDs, LEDs using quantum dot technology, andLED arrays. Alternatively, the light sources can include white light,halogen lamp, lasers, solid state laser, laser diode, micro-wire laser,diode solid state lasers (DSSL), vertical-cavity surface-emitting lasers(VCSEL), thin-film electroluminescent devices (TFELD), filament lamps,arc lamps, gas lamps, and fluorescent tubes. Light sources can have highradiance, such as lasers, or low radiance, such as LEDs. Radiance refersto light emitted and can be measured in units of watts per centimetersquared per steradian. Lasers have high radiance since they emit lightin substantially a single direction. LEDs have low radiance since theytypically emit light into 2 pi steradians. The different types of LEDsmentioned above can have a medium to high radiance.

The term “detector” as used herein refers to any component, portionthereof, or system of components that can detect light including acharged coupled device (CCD), back-side thin-cooled CCD, front-sideilluminated CCD, a CCD array, a photodiode, a photodiode array, aphoto-multiplier tube (PMT), a PMT array, complimentary metal-oxidesemiconductor (CMOS) sensors, CMOS arrays, a charge-injection device(CID), CID arrays, etc. The detector can be adapted to relay informationto a data collection device for storage, correlation, and/ormanipulation of data, for example, a computer, or other signalprocessing system.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A method for processing biological or chemicalsamples comprising: positioning a single standard microtiter plate on aplurality of reaction vessel receiving elements of a thermal cycler;independently heating and cooling the plurality of reaction vesselreceiving elements with a plurality of thermoelectric cooling devices(TEC); sealing the area below the plurality of reaction vessel receivingelements with a drip pan, a gasket, and a clamp, wherein the gasket hasa convex portion and the clamp provides a lateral force to compress thegasket between the reaction vessel receiving elements to form anairtight seal between each of the plurality of reaction vessel receivingelements; and thermally isolating adjacent reaction vessel receivingelements by constructing the gasket from a non-thermally conductingmaterial.
 2. The method of claim 1, further comprising: annealingsamples in a first portion of the microtiter plate at a first annealingtemperature by cooling a first reaction vessel receiving element; andannealing samples in a second portion of the microtiter plate at asecond annealing temperature by cooling a second reaction vesselreceiving element, wherein the second annealing temperature is not equalto the first annealing temperature.
 3. The method of claim 1, furthercomprising: elongating samples in a first portion of the microtiterplate at a first elongation temperature by heating a first reactionvessel receiving element; and elongating samples in a second portion ofthe microtiter plate at a second elongation temperature by heating asecond reaction vessel receiving element, wherein the second elongationtemperature is not equal to the first elongation temperature.
 4. Themethod of claim 1, further comprising: repeating for a first number ofcycles at least one of the steps of denaturing, annealing, andelongating samples in a first portion of the microtiter platecorresponding to a first reaction vessel receiving element; andrepeating for a second number of cycles at least one of the steps ofdenaturing, annealing, and elongating samples in a second portion of themicrotiter plate corresponding to a second reaction vessel receivingelement, wherein the first number of cycles is not equal to the secondnumber of cycles.
 5. The method of claim 1, wherein a rate of cooling ofa first reaction vessel receiving element is not equal to the rate ofcooling of a second reaction vessel receiving element.
 6. The method ofclaim 1, wherein a rate of heating of a first reaction vessel receivingelement is not equal to the rate of heating of a second reaction vesselreceiving element.
 7. The method of claim 1, wherein the samples in afirst reaction vessel receiving element have a different volume than thesamples in a second reaction vessel receiving element.
 8. The method ofclaim 1, wherein a first reaction vessel receiving element is kept at afirst residence time for annealing samples and a second reaction vesselreceiving element is kept at a second residence time for annealingsamples.
 9. The method of claim 1, wherein a first reaction vesselreceiving element is kept at a first residence time for elongatingsamples and a second reaction vessel receiving element is kept at asecond residence time for elongating samples.